TRAIL is a candidate for cancer therapy that selectively induces apoptosis in transformed/tumor cells but not in normal cells. However, the mechanism for the tumor selectivity of TRAIL-induced apoptosis is poorly understood. FLICE Inhibitory Protein (c-FLIP) is a regulator of programmed cell death. Recently, we have demonstrated that full-length c-FLIPL interacts with one of the TRAIL receptors (DR5) in the absence of ligand to prevent apoptosis. Upon induction of apoptosis by TRAIL, the major c-FLIP variants at the TRAIL DISC (Death Inducing Signaling Complex) are c-FLIPp43 (product of caspase cleavage) and c-FLIPs. Our studies further indicate that elimination of c-FLIPL by proteolytic cleavage is an early step in TRAIL-induced apoptosis and that c-FLIPL, c-FLIPs and c-FLIPp43 have distinct roles in mediating TRAIL function. In support of this hypothesis, we have observed that c-FLIPL, but not c-FLIPp43, promotes activation of the c-Akt survival pathway. The overall goal of this application is to elucidate the molecular mechanisms by which the c-FLIP variants regulate TRAIL-induced apoptosis and the distinct roles of the c-FLIP variants in the tumor selectivity of TRAIL-induced apoptosis in vitro and in an in vivo model for Bcr-Abl-induced chronic myeloid leukemia. In Aim 1, we will determine the specific functions of the c-FLIP variants and the role of caspase cleavage of c-FLIPL in TRAIL-induced apoptosis. This will be achieved in vitro by using c-FLIP (-/-) embryonic fibroblasts that stably express each of the c-FLIP variants, and by siRNAs that specifically target the c-FLIP variants. Furthermore, we will determine the molecular mechanism by which the c-FLIP variants regulate TRAIL-induced apoptosis by characterizing the distinct signaling complexes that they regulate. In Aim 2, we will delineate the novel role of the c-Akt survival pathway in mediating the anti-apoptotic activity of c-FLIP. We will also investigate the molecular mechanism by which c-FLIP induces Akt activation. In Aim 3, we will determine the specific roles of the c-FLIP variants in mediating selective tumor death by TRAIL and their utilization as a predictive marker for TRAIL-therapy. These studies will be performed in vitro using Bcr-Abl transformed cells and in an in vivo model for Bcr-Abl-induced leukemia.